【作者】 曹娟；

【导师】 韩召军；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2004， 硕士

【摘要】 棉蚜（Aphis gossypii Glover）是世界性分布的重大农业害虫，生产上主要依赖于化学防治，这导致了棉蚜对多种杀虫剂产生了不同程度的抗药性。解毒代谢酶活力增强和杀虫剂靶标敏感性下降是棉蚜抗药性形成的重要机制，其中乙酰胆碱酯酶敏感性下降是棉蚜对有机磷和氨基甲酸酯类杀虫剂产生抗性的重要机制，而羧酸酯酶也在棉蚜对有机磷类杀虫剂的抗性中扮演着重要角色。因此，本文就抗性和敏感品系棉蚜乙酰胆碱酯酶和酯酶进行了一系列的比较研究。 1 棉蚜乙酰胆碱酯酶粗酶液稳定性研究 在抗药性研究中，经常利用粗酶液研究乙酰胆碱酯酶的生理毒理学特性。但棉蚜乙酰胆碱酯酶极易失活，因此对棉蚜乙酰胆碱酯酶粗酶液的稳定性进行研究，有助于获得可靠的结果和结论。本研究通过比较不同温度、以及加入甘油和牛血清白蛋白对酶活力的影响，分析了影响棉蚜乙酰胆碱酯酶粗酶液稳定性的重要因子。 研究结果显示，在4种不同温度下（-20℃、4℃、15℃、25℃），随着环境温度的升高，棉蚜乙酰胆碱酯酶活性丧失速度加快，-20℃保存时，乙酰胆碱酯酶最为稳定，酶活力可以维持较长的时间。而在25℃下，乙酰胆碱酯酶最不稳定，活性丧失很快。一定浓度的甘油可提高乙酰胆碱酯酶在低温保存时的稳定性，但加入稳定剂牛血清白蛋白，对粗酶液中酶的稳定性并不能产生显著效果。由此认为，研究棉蚜乙酰胆碱酯酶的粗酶液，应保存在-20℃以下，并应加入一定量的甘油，以稳定酶活性，延长保存时间。 此外，本研究还发现，无论如何处理，酶液制备后的一段时间内，酶活力均呈现下降趋势，且不同品系棉蚜制备的乙酰胆碱酯酶粗酶液，其活力随时间的变化情况明显不同。因此建议，进行棉蚜乙酰胆碱酯酶活力的比较研究时，除需在-20℃下长期保存以外，还需要控制酶液保存时间的一致性。 2 不同抗性品系棉蚜的乙酰胆碱酯酶和羧酸酯酶比较 采用改进的棉蚜饲养方法，建立了不同抗性品系的棉蚜。通过测定来自不同地区（南京、北京和安阳）的抗性品系棉蚜乙酰胆碱酯酶对3种药剂（甲胺磷、乙酰甲胺磷和抗蚜威）的敏感性(I₅₀和K_i)，发现安阳品系棉蚜乙酰胆碱酯酶对杀虫剂的敏感性显著下降，而南京、北京品系则接近于敏感品系，但这3个抗性品系的全酯酶和羧抗性和敏感性棉蚜的乙酞胆碱酷酶和酷酶的比较研究酸醋酶的活性均显著高于敏感品系。分析认为，低抗水平的南京、北京品系只存在代谢杭性，还未产生靶标抗性，而高杭的安阳品系，代谢抗性和靶标抗性均存在，导致了高水平抗性的形成。 利用3种杀虫剂对不同品系棉蚜乙酞胆碱醋酶所做的抑制曲线，其结果与与和龙的测定结果完全一致，但并没有发现因2种（或抗、感）乙酸胆碱醋酶的存在而出现的“平台”。由此认为，利用抑制毒理曲线的“平台”作为鉴别有无2种酶的存在，并不完全可靠。 此外，以a一乙酸蔡醋为底物时，棉蚜欺酸醋酶的活性显著高于以日一乙酸蔡醋为底物时的活性，但不同品系没有发现顺序差异，说明棉蚜发酸醋酶对底物的空间构型可能很敏感。3不同品系棉蚜乙酞胆碱醋酶和梭酸醋酶亚细胞分布特征 通过差速离心法分析杭性品系和敏感品系棉蚜乙酞胆碱醋酶和数酸醋酶的亚细胞分布，结果显示杭性品系和敏感品系棉蚜的乙酞胆碱醋酶和发酸醋酶的亚细胞分布均存在显著差异。未用Tr 1 tonX一100处理时，抗性品系乙酞胆碱醋酶活性主要分布于细胞核、细胞碎片及线粒体层中，而敏感品系乙酸胆碱醋酶活性主要分布于微粒体层中，用Tr 1 tonx一100处理后，杭性品系和敏感品系乙酞胆碱醋酶均主要集中到上清液中。由于微粒体可能包含了合成和分泌该酶的内质网和囊泡，而细胞核、细胞碎片及线粒体的酶活性更能体现酶的膜结合状态，因此认为杭性品系棉蚜的乙酞胆碱醋酶主要以膜结合蛋白形式存在，而敏感品系的主要以游离状态存在。 抗性品系欺酸醋酶活性主要分布在细胞核、细胞碎片及线粒体层中，敏感品系狡酸醋酶活性则主要集中在上清液中，并且杭性品系各亚细胞组分的活性均显著高于敏感品系。 显然，杭性品系和敏感品系的乙酞胆碱醋酶和致酸醋酶不仅存在着杭性水平的差异，还存在着亚细胞分布上的差异，杭性品系的乙酸胆碱醋酶和戎酸醋酶具有显著的亲膜特性。这种亲膜特性是否与抗药性直接相关，尚需要做进一步研究，但这一研究结果表明，做抗、感棉蚜酶活力比较研究时，必须加入Tr 1 tonx一100等表面活性剂，才能保证酶提取的一致性。 上述研究成果对于今后准确分析棉蚜乙酞胆碱醋酶和发酸醋酶的活力和特性，以及对于不同抗性水平棉蚜的治理措施的设计，均具有重要的意义。更多还原

【Abstract】 As a w orldwide-distributed p est, c otton a phid, Aphis g ossypii G lover, a ttracts 1 ots o f attention. The rapid development of resistance to insecticides has brought difficulty in the field control of this important pest. Acetylcholinesterase (AChE) is a target for organophosphate (OP) and carbamate insecticides. Insecticides resistance due to the insensitivity of acetylcholinesterase to the inhibition by these two groups of insecticides has been found in many insects including cotton aphid. Carboxylesterase (CarE) also plays an important role in organophosphate resistance of cotton aphid. In this paper, comparing studies on AChE and CarE from resistant and susceptible strains have been performed, including their stability, toxicology and sub-cellular distribution.1 Study of stability of crude acetylcholinesterase from cotton aphidThe crude preparation of AChE is usually used in pest resistance study. However, AChE is not stable and easy to breakdown. Studies on the factors affecting the stability of the enzyme would be helpful for getting correct results in the further researches. So, series of experiments were carried out to demonstrate if and how temperature, glycerol and bovine serum albumin (BSA) affect the stability of the enzyme.The results showed that the lower temperature when crude AChE preparation was stored at, the more stable it was. Glycerol could improve its stability effectively when AChE was stored at -20C. But BSA couldn’t significantly improve the stability of the enzyme in crude preparations when stored at higher temperature. It was suggested that crude AChE preparations should be stored at -20C and with some glycerol.AChE was also found having the tendency to lose its activity gradually short after it was extracted, and the stability of the crude AChE prepared from different strains, resistant and susceptible, were apparently different. So, the enzyme preparations stored for similar time should be used for comparing study of their activity.2 Comparison of acetylcholinesterase and carboxylesterase from different aphid strainsWith improved caging method of Li fei (2001), four laboratory strains of cotton aphid were set up. A susceptible strain 171B was introduced from Rothamsted Experiment Strains, UK. Three resistant strains were bred from the aphids collected from different districts in China, Nanjing, Beijing and Anyang. With the AChE preparations from these 4 strains, theI50 and Ki of two OP and one carbamate insecticides were tested. Compared with that from 171B, the AChE from Anyang strain was found apparently insensitive to all the insecticides tested. But the sensitivity of AChE from Nanjing and Beijing strains were similar as that from 171B. However, the esterase (total esterase and carboxylesterase) activity increased dramatically in all the three strains. So, it could be deduced that pesticide resistance in Nanjing and Beijing strains only attributed to increased pesticide sequestration or detoxification by esterase, while the resistance in Anyang strain attributed to both increased esterase activity and reduced sensitivity of AChE.The inhibition curve test was also carried out with the same insecticides and cotton aphid strains. The results showed the similar insensitivity of the AChE from different strains. But no flat on the curves was observed as usually expected when different enzymes present. So, it was thought it is not reliable to identify different enzymes with the flat on inhibition curves.3 Subcellular distribution of acetylcholinesterase and carboxylesterase from different aphids strainsWith differential centrifugation, subcellular distribution of AChE and CarE from susceptible strain (171B) and resistance strain (Anyang strain) were studied. Results showed that both AChE and CarE from susceptible strain and resistance strain were differently sub-cellular distributed. AChE activities in resistant strain were mainly distributed in cell nucleus, cell debris and mitochondrion. While AChE activities in susceptible strain were mainly更多还原